Thermodynamic Origins of Sequence-Recognition in Ligand-DNA Interactions

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9728720 Graves The focus of this research is to gain insight into the mechanisms through which small molecules and proteins exert sequence-selective binding to nucleic acids. Efforts will be directed towards correlating the thermodynamic properties associated with these interactions with both DNA sequence and ligand structure. Recent studies have demonstrated the DNA binding enthalpies and entropies observed for actinomycin D-DNA interactions to be markedly influenced not only by the base-sequence at the intercalation site but also by the bases that flank the intercalation site. These observations will be extended to explore the effects of subtle changes in the DNA base sequences that flank the ligand binding site and their relationship to changes in the thermodynamic binding mechanism(s). Analyses of thermodynamic binding properties will be coupled to examination of the concomitant ligand-DNA structures resulting from interactions of ligands with specific DNA base sequences providing linkage between the thermodynamic mechanisms associated with complex formation and the structural geometry of the ligand-DNA complex. Various biophysical methods including absorption spectroscopy and isothermal microtitration calorimetry will be used to examine the interactions of actinomycin D with oligonucleotides that have been designed specifically to harbor a single centrally located actinomycin D binding site and varied neighboring and next-neighboring flanking base sequences. The binding site sequence and bases which flank the binding site will be systematically changed to determine their influence on the thermodynamic mechanism of complex formation. From these studies, insight into the thermodynamic origins of DNA sequence-recognition will be discerned. Structural details of the ligand-DNA complex will be examined by high-resolution nuclear magnetic resonance methods and correlated with the thermodynamic data to provide a novel paradigm for the "thermodynamic origins" of sequence-recognition. Mol ecular recognition is one of the central problems in understanding the mechanistic processes that govern a wide variety of biochemical and molecular biological events that take place in the cell. Hence, an understanding of the forces that govern the sequence specific interactions of small molecules and proteins with DNA is pivotal towards an overall comprehension of basic biochemical processes such as replication, transcription, recombination, and DNA repair. The focus of this research is to determine and characterize the chemical and/or structural determinants on both the target DNA and ligand responsible for sequence-selective interactions.